Академический Документы
Профессиональный Документы
Культура Документы
Behavioral/Cognitive
Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area.
Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the
regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic
mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed
knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was
controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic
activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during
wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states.
Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased
NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but
not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance
of NREM sleep.
Key words: ablation; cell fate; channelrhodopsin2; hypothalamus; optogenetics; REM sleep
Introduction
Melanin-concentrating hormone (MCH)-producing neurons
are coexpressed with orexin/hypocretin-producing neurons in
Received Dec. 18, 2013; revised April 2, 2014; accepted April 8, 2014.
Author contributions: A.Y. designed research; T.T., T.U., S.T., A.T., and A.Y. performed research; K.F.T., H.H., and
A.Y. contributed unpublished reagents/analytic tools; T.T., T.U., S.T., A.I., T.S.K., A.T., and A.Y. analyzed data; T.T.,
K.F.T., T.S.K., A.T., and A.Y. wrote the paper.
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas Mesoscopic Neurocircuitry (23115103), a Grant-in-Aid for Scientific Research (B) (23300142; A.Y.), a Grant-in-Aid for Scientific Research
(C) (22500830; A.T.), and a Grant-in-Aid for Young Scientist (A) (23680042; K.T.) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan; by the Prescursor Research for Embryonic Science and Technology
(PRESTO) program from Japan Science and Technology Agency (A.Y.); by Takeda Science Foundation and Kanae
Foundation by a Japan Society for Promotion of Science postdoctoral fellowship (T.T. and S.T.); and by National
Institutes of Health Grant R01 NS077408 (T.K.). We thank Dr. Y. Ootsuka and Dr. K. Matsui for assisting in data
analyses. We thank C. Saito, K. Nishimura, Y. Esaki (Nonprofit Organization Biotechnology Research and Development), and S. Nishioka (Nonprofit Organization Biotechnology Research and Development) for technical assistance.
The authors declare no competing financial interests.
This article is freely available online through the J Neurosci Author Open Choice option.
Correspondence should be addressed to Akihiro Yamanaka, PhD, Department of Neuroscience II,
Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan. E-mail:
yamank@riem.nagoya-u.ac.jp.
DOI:10.1523/JNEUROSCI.5344-13.2014
Copyright 2014 the authors 0270-6474/14/336896-14$15.00/0
the lateral hypothalamic area (LHA) but are more numerous and
extend further rostrocaudally (Bittencourt et al., 1992; Peyron et
al., 1998; Elias et al., 2008). MCH neurons project widely
throughout the brain and densely innervate the cholinergic and
monoaminergic arousal centers (Bittencourt et al., 1992). MCH
neurons have been reported to be GABAergic (Del Cid-Pellitero
and Jones, 2012). While MCH receptor-1 (MCHR1; the only
receptor found in rodents) binding activates Gq, Gi, and Go subunits (Hawes et al., 2000), the major effect of MCHR1 binding is
to decrease cAMP levels (Chambers et al., 1999; Lembo et al.,
1999) and cellular electrophysiological studies have revealed presynaptic and postsynaptic inhibitory effects of MCH (Gao and
van den Pol, 2001; Wu et al., 2009). Thus, MCH neurons exert
neuroinhibitory effects on downstream targets.
MCH has been implicated in several functions, including
feeding, energy balance, and locomotor activity (Shimada et al.,
1998; Asakawa et al., 2002; Marsh et al., 2002; Semjonous et al.,
2009); its involvement in sleep and wakefulness has also been
extensively studied. Intracerebroventricular injections of MCH
early in the dark period increased rapid eye movement (REM)
sleep in a dose-dependent manner (Verret et al., 2003); some
software version 3 (Kissei Comtec). Blue (475 17.5 nm) and green light
(542 13.5 nm) was generated by the SPECTRA light engine and applied
through plastic optical fibers bilaterally inserted 1 mm above the LHA.
An optical swivel (COME2, Lucir) was used for unrestricted in vivo photo
illumination. The power intensities of blue and green light at the tip of
the plastic fiber optics (0.5 mm diameter) were 25.8 and 33.4 mW/mm 2,
respectively, as measured by a power meter (VEGA, Ophir Optronics).
Each animals behavior was monitored through a CCD video camera and
recorded on a computer synchronized with EEG and EMG recordings
using the SleepSign video option system (Kissei Comtec). The infrared
activity monitor was a sensor mounted on top of the cage to measure
locomotor activity. The sensors output signals (representing the magnitude of each animals movement) were digitally converted and transferred to a computer.
MCH-tTA; TetO DTA bigenic mouse surgery. MCH-tTA; TetO DTA
bigenic mice were housed under controlled lighting (12 h light/dark
cycle; lights on from 7:00 to 19:00) and temperature (22C) conditions.
Food and water were available ad libitum. Doxycycline-containing chow
(Dox chow) was made by adding 10% doxycycline (Dox) powder (Kyoritsu Seiyaku) to normal chow (Labo MR Stock, Nosan) at a final concentration of 100 mg/kg. Labo MR stock was provided during the
Dox() period (from 10 weeks of age). Mating pairs of MCH-tTA mice
and TetO DTA mice were fed with Dox-containing chow [Dox() condition] from the day of mating. During the prenatal and early postnatal
periods, Dox was supplied via maternal circulation or lactation, respectively. After weaning, MCH-tTA; TetO DTA mice were fed with Dox()
chow until the day of the experiment.
MCH-tTA; TetO DTA bigenic mice (8 weeks of age) were anesthetized
with an intraperitoneal injection of a drug mixture containing ketamine
(75 mg/kg) and medetomidine (1 mg/kg), placed in a stereotaxic device,
and chronically implanted with EEG and EMG electrodes for polysomnographic recording of sleep and wakefulness states. Two stainless-steel
screws (diameter, 1.0 mm) were implanted into the skull over the left
cerebral hemisphere (frontal: 1.5 mm lateral to midline, 1.0 mm anterior
to bregma; parietal: 1.5 mm lateral to midline, 1.0 mm anterior to
lambda) according to the atlas of Franklin and Paxinos (1997). A third
screw was placed over the frontal bone to serve as a ground electrode.
EMG activity was monitored using stainless-steel Teflon-coated wires
inserted bilaterally into the neck muscles. All electrodes were attached to
a microconnector, and the entire assembly was fixed to the skull with
dental cement. Mice were injected with indomethacin (1 mg/kg, i.p.) and
penicillin G (200 U/kg, i.m.) immediately after surgery. All procedures
were performed on a heating pad, and mice were allowed to recover for
10 14 d before being transferred to sleep recording chambers.
Vigilance state determination. Polysomnographic recordings were automatically scored offline as wakefulness, NREM sleep, or REM sleep by
SleepSign, in 4 s epochs (optogenetics experiments) or 10 s epochs (ablation experiments), according to standard criteria (Tobler et al., 1997;
Yamanaka et al., 2002). All vigilance state classifications assigned by
SleepSign were examined visually and corrected if necessary. The same
individual, blinded to genotype and experimental condition, scored all
EEG/EMG recordings. Spectral analysis of the EEG was performed by fast
Fourier transform (FFT; sampled at 128 Hz). This analysis yielded a
power spectral profile over a 0 25 Hz window with a 1 Hz resolution
divided into delta (15 Hz), theta (6 10 Hz), alpha (10 13 Hz), and beta
(1325 Hz) waves.
Statistical analysis. Data were analyzed by paired t test, unpaired t test,
one-way ANOVA, two-way ANOVA, or nonparametric KruskalWallis
test (as appropriate for the parameters examined) using KaleidaGraph
4.0 software (Hulinks). When appropriate, ANOVA tests were followed
by post hoc analysis of significance using Fishers protected least significant difference test or Bonferronis test. p values 0.05 were considered
statistically significant.
Results
Specific activation of MCH neurons using ChR2
MCH neurons in the brains of MCH-tTA; TetO ChR2 bigenic
mice specifically expressed ChR2 (Fig. 1A) as confirmed by
double-labeled immunohistochemistry. An anti-GFP antibody
Figure 1. Specific activation of MCH neurons. A, Schematic showing generation of bigenic MCH-tTA; TetO ChR2 mice. P, Minimal
promoter. B, C, Immunohistochemical analyses revealed that ChR2 is specifically expressed in MCH neurons in the MCH-tTA; TetO
ChR2 bigenic mouse brain. B, MCH-immunoreactive (MCH-IR) neurons (left, Alexa594, red) and ChR2::EYFP-immunoreactive
(YFP-IR) neurons (middle, Alexa488, green) located in the LHA and the zona incerta. Right, Merged image shows specific expression of ChR2 in MCH neurons. Bottom row represents higher magnifications of the regions enclosed by the squares in the top row.
Scale bars: (in top, right) top row, 200 m; (in bottom, right) bottom row, 100 m. C, Confocal microscopic image of the region
indicated by the squares in B, bottom row. Scale bar, 20 m. DH, Slice patch-clamp recordings from MCH neurons. D, Photocurrent induced by blue light (475 17.5 nm, 2.5 mW, 2 s; Vh 60 mV, TTX 1 M). E, Bar graph summarizing the data obtained
from D. F, Current-clamp mode recording, blue light pulses (2.5 mW, 10 ms, 10 Hz) generated action potentials in MCH neurons. G,
Loose cell-attached recording from MCH neurons. Blue light pulses (2.5 mW, 10 ms, 10 Hz) for 1 min every 5 min generated action
potentials. H, Summary data from G. Values represent means SEM. *p 0.05 versus EGFP-expressing MCH neurons.
Table 1. Vigilance state parameters recorded from MCH-tTA; TetO ChR2 bigenic mice, monogenic littermate mice, and MCH-tTA; TetO ArchT bigenic mice under basal
conditions
Wakefulness
REM sleep
NREM sleep
24 h
Total time (min)
Episode duration (s)
Number of episodes (counts)
Light period
Total time (min)
Episode duration (s)
Number of episodes (counts)
Dark period
Total time (min)
Episode duration (s)
Number of episodes (counts)
Control
MCH-tTA;
TetO ChR2
MCH-tTA;
TetO ArchT
Control
MCH-tTA;
TetO ChR2
MCH-tTA;
TetO ArchT
Control
MCH-tTA;
TetO ChR2
MCH-tTA;
TetO ArchT
792.5 40.7
479.1 91.7
118.0 4.6
734.5 30.3
479.3 81.9
108.1 8.1
755.9 14.8
546.7 86.9
131.4 11.4
61.5 6.8
65.8 4.8
52.6 3.4
81.2 9.3
75.1 1.6
48.7 4.6
80.8 10.6
71.7 4.7
59.4 6.6
586.0 10.6
281.6 26.2
133.3 5.1
624.2 25.1
251.6 12.1
127.0 6.8
603.2 12.3
251.2 13.2
148.2 10.5
232.4 14.7
212.3 25.9
79.4 9.0
210.2 9.2
167.3 13.7
80.9 10.4
204.9 5.9
169.8 30.7
95.2 5.7
50.5 8.1
71.5 7.6
39.5 4.3
62.5 5.0
83.7 3.7
41.3 3.8
68.1 8.1
79.8 3.1
48.2 5.1
437.1 16.5
344.8 42.8
90.4 9.8
447.3 12.2
285.7 33.9
97.7 8.9
446.9 6.2
269.0 15.4
108.6 6.1
551.0 43.9
745.8 204.8
38.6 8.2
524.4 37.6
791.3 153.4
27.3 5.9
551.0 16.8
923.6 147.3
36.2 7.2
12.7 2.9
60.2 3.4
13.1 3.0
18.7 5.7
66.4 2.9
7.4 2.0
12.7 3.4
63.6 9.7
11.2 1.7
156.4 41.3
218.3 12.6
42.9 9.4
176.9 32.2
217.4 22.4
29.3 6.5
156.3 14.9
233.4 24.6
39.6 6.4
Figure 2. In vivo optogenetic activation of MCH neurons increased time spent in REM sleep. A, F, Bar graphs illustrating the time in each vigilance state during the light period (9:00 16:00) in
(A) MCH-tTA; TetO ChR2 bigenic mice and (F ) MCH-tTA monogenic mice and TetO ChR2 monogenic mice. Blue light pulses (475 17.5 nm, 25.8 mW/mm 2, 10 ms, 10 Hz for 3 h from 10:00 to 13:00)
were applied. Blue background indicates the period of illumination. B, G, Bar graphs summarizing the data from A and F. The time spent in each vigilance state during illumination for 3 h is
summarized. Black bars, No light illumination; blue bars, blue light pulse illumination. C, H, Mean episode duration. D, I, Number of episodes. E, J, Bar graph summarizing the time in each vigilance
state for 1 h after cessation of illumination (from 13:00 to 14:00) in MCH-tTA; TetO ChR2 bigenic mice (E) and MCH-tTA monogenic mice and TetO ChR2 monogenic mice (J ). W, Wakefulness; R, REM
sleep; NR, NREM sleep. Values are represented as means SEM. *p 0.05 versus basal (no illumination).
by the increased number of episodes of all states. After stimulation of MCH neurons ceased, NREM sleep was significantly increased with decreases in wakefulness and REM sleep (Fig. 2E),
suggesting a compensatory rebound in NREM sleep.
Blue light illumination (475 17.5 nm, 25.8 mW/mm 2, 10
ms, 10 Hz) had no effect on sleep/wakefulness in monogenic
MCH-tTA or TetO ChR2 mice (Fig. 2F ). Neither the total time
(Fig. 2G), nor mean episode duration (Fig. 2H ), nor the number
of episodes (Fig. 2I ) during illumination for 3 h, nor the total
time after illumination (Fig. 2J ) of each vigilance state were significantly affected by blue light pulse.
Does activation of MCH neurons induce physiological
REM sleep?
Although activation of MCH neurons increased the time spent in
REM sleep as determined from EEG and EMG recordings in
freely moving mice, it was unclear whether this state met criteria
for physiological REM sleep other than those based on EEG and
EMG recordings. To evaluate this further, eye and whisker movements during activation of MCH neurons were visually analyzed.
To monitor eye and whisker movements during MCH neuron
stimulation, mice were affixed to a stereotaxic frame via a plastic
U-shape frame fixed onto the skull. After a week adaptation and
training to these conditions, mice spontaneously cycle between
wakefulness and sleep. While a mouse was in spontaneous NREM
sleep, MCH neurons were activated and the EEG and EMG indicated signs of REM sleep shortly after initiation of stimulation
(475 17.5 nm, 25.8 mW/mm 2, 10 ms, 10 Hz). During MCH
neuron activation-induced REM sleep, the eyes and whiskers
moved intermittently, similar to that observed during spontaneously occurring REM sleep. These results suggest that MCH neuron activation-induced REM sleep is indistinguishable from
physiologically occurring REM sleep in terms of EEG, EMG, and
eye and whisker movement indicators.
Activation of MCH neurons during NREM sleep but not
during wakefulness induces REM sleep
The results presented in Figure 2 clearly demonstrate that REM
sleep can be induced by MCH neuron stimulation during NREM
sleep. To determine whether activation of MCH neurons during
wakefulness also induces a transition to REM sleep, MCH neurons were intermittently activated for 1 min (475 17.5 nm, 25.8
mW/mm 2, 10 ms, 10 Hz) every 5 min from 20:00 (the onset time
of the active dark period) to 16:46 (the second half of the inactive
light period). MCH-tTA; TetO ChR2 mice without light illumination were used as controls. Figure 3A shows a representative
hypnogram from MCH-tTA; TetO ChR2 mice under basal conditions (without light illumination). In comparison, the hypnogram of MCH-tTA; TetO ChR2 mice with blue light illumination
(Fig. 3B) shows frequent transitions to REM sleep in conjunction
with light illumination. However, transitions to REM sleep were
restricted to epochs during which mice were in NREM sleep.
Activation of MCH neurons in NREM sleep immediately decreased EEG delta power and increased theta power, suggesting a
transition to REM sleep (Fig. 3C,D). Mean REM sleep latency
calculated from the initiation of blue light illumination was
12.4 1.2 s (n 129). REM sleep was often terminated when
blue light illumination ended. In contrast, activation of MCH
neurons did not induce a transition to REM sleep when mice were
awake (Fig. 3 E, F ). EEG and EMG were indistinguishable from
before MCH neuron activation.
Figure 3GJ presents the percentage of transitions during and
after 1 min MCH neuron activation during the dark period (Fig.
Figure 3. Acute activation of MCH neurons induces transitions from NREM sleep to REM sleep. A, B, Representative hypnograms of (A) no illumination and (B) blue light illumination for 1 min
every 5 min (25.8 mW/mm 2, 10 ms, 10 Hz) from 20:00 to 16:46. The upper panel is the dark period (20:00 8:00); lower panel is the light period (8:00 20:00); blue bars indicate 1 min periods of
blue illumination. C, E, Representative traces for EEG (upper trace) and EMG (lower trace). Blue pulses were applied during NREM sleep (C) or wakefulness (E). D, F, EEG power spectra corresponding
to C and E. GJ, Graphs show percentage of animals in each vigilance state during blue pulse illumination. G, H, Dark period. I, J, Light period. Blue light illumination occurred during a waking period
in G and I and during NREM sleep in H and J. The blue lines above each graph indicate 1 min blue light illumination pulses. K, Power spectral analysis of EEG in the light period (8:00 16:46) without
illumination (black) and with illumination (blue). L, The average EEG power densities in the delta, theta, alpha, and beta wave bands in the light period (8:00 16:46). W, Wakefulness; R, REM sleep;
NR, NREM sleep; LP, light period; DP, dark period. Values are represented as means SEM. *p 0.05 versus no illumination.
Figure 4. In vivo optogenetic inhibition of MCH neurons has no effect on vigilance states. A, Schematic shows generation of bigenic MCH-tTA; TetO ArchT mice. P, Minimal promoter. B, C,
Immunohistochemical analyses revealed that ArchT is specifically expressed in MCH neurons in the MCH-tTA; TetO ArchT bigenic mouse brain. B, MCH-immunoreactive (MCH-IR) neurons (left, red;
Alexa594) and ArchT::GFP-immunoreactive (GFP-IR) neurons (middle, green; Alexa488) located in the LHA and the zona incerta. Right, Merged image shows specific expression of ArchT in MCH
neurons. Bottom row are higher magnifications of the regions enclosed by the squares in the top row. Scale bars: (in top, right) top row, 200 m; (in bottom, right) bottom row, 100 m. C, Confocal
microscopic image of the region indicated by the squares in B, bottom row. Scale bar, 20 m. D, E, Slice patch-clamp recordings from MCH neurons. D, Upper trace, Current-clamp recording from
MCH neuron. Lower trace, Injected current through the recording electrode (0.2 Hz, 140 pA, 100 ms) to generate action potentials. Green light illumination (549 7.5 nm, 0.9 mW, 1 min) inhibited
firing elicited by current injection. E, Bar graph indicating the firing probability calculated from the data in D (n 7). Firing probability is normalized to the 1 min before light illumination (Pre). Green
light was applied for the duration indicated by the green bars. Values represent means SEM. *p 0.05 versus Pre. F, Green light illumination (542 13.5 nm, 33.4 mW/mm 2, 3 h from 13:00
to 16:00) in the light period. GI, Bar graphs indicating the total time in each vigilance state (G), episode duration (H ), and number of episodes (I ) during green light illumination. W, Wakefulness;
R, REM sleep; NR, NREM sleep. Values are represented as means SEM. *p 0.05 versus basal (no illumination).
During the light (inactive) period, green light (542 13.5 nm,
33.4 mW/mm 2) was illuminated through fiber optics onto the
hypothalamus of MCH-tTA; TetO ArchT mice for 3 h from 13:00
to 16:00 using the same methods used for activation of MCH
neurons. MCH-tTA; TetO ArchT mice without illumination were
used as controls. Optogenetic inhibition of MCH neurons had no
significant effect on time spent in any vigilance state (Fig. 4 F, G).
In the control condition, the time spent in wakefulness, REM
sleep, and NREM sleep over the 3 h period was 47.7 3.5 min
(n 6), 18.6 2.9 min (n 6), and 113.6 2.1 min (n 6),
respectively. The time spent in wakefulness, REM sleep, and
NREM sleep during green light illumination was 46.6 5.9 min
(n 6, p 0.88, NS, paired t test), 14.5 3.3 min (n 6, p
0.054, NS, paired t test), and 118.9 5.5 min (n 6, p 0.41,
NS, paired t test), respectively. Inhibition of MCH neurons also
had no effect on episode duration (Fig. 4H ) or the number of
episodes (Fig. 4I ). As indicated in Table 1, ArchT expression in
MCH neurons had no effect on basal sleep/wakefulness patterns
(total time and episode duration of each state). These results
suggest that silencing of MCH neurons has little effect on the
initiation and maintenance of REM sleep.
Ablation of MCH neurons decreases time spent in
NREM sleep
Inhibition of MCH neurons using optogenetics had little effect
on sleep/wakefulness. However, due to the technical limitations
of optogenetic inhibition, particularly for long periods of inhibition, we wanted to induce a chronic loss of function to further
evaluate the role of MCH neurons in sleep/wakefulness regulation. Thus, we used the tet-off system mice to specifically ablate
MCH neurons. MCH-tTA mice were bred with TetO DTA mice
to generate MCH-tTA; TetO DTA bigenic mice (Fig. 5A). In these
mice, DTA expression is restricted to MCH neurons and DTA
induces cell death by inhibiting protein synthesis. The timing of
DTA expression is controlled by the presence or absence of Dox
in the chow. In the presence of DTA, tTA loses its ability to bind
the TetO sequence (Fig. 5B). However, in the absence of Dox, tTA
induces DTA expression. Here, MCH-tTA; TetO DTA mice were
fed with chow containing Dox (100 mg/kg) until 10 weeks of age.
Then, Dox() chow was replaced with Dox() chow, the timing
of which was defined as Dox() 0 week (Fig. 5C).
MCH neuron-specific and temporally controlled ablation was
confirmed by immunohistochemical studies using an anti-MCH
antibody. The number of MCH-immunoreactive neurons was
counted in C57BL/6J wild-type mice, in TetO DTA monogenic
mice at 10 weeks of age, and in MCH-tTA; TetO DTA bigenic
mice at DOX() 0, 1, 2, 3, and 4 weeks. At Dox() 0 week, the
number of MCH neurons was 1028 33 (n 3). This was
comparable to the number of MCH neurons in the C57BL/6J
wild-type mice (1076 61, n 4, p 1, KruskalWallis) and in
the TetO DTA monogenic mice (1031 65, n 3, p 1,
KruskalWallis). Meanwhile, the number of MCH neurons dramatically decreased after Dox removal (Fig. 5 D, E). The number
of MCH neurons at Dox() 1, 2, 3, and 4 weeks was 600 52
(58.3%, n 3, p 0.001, KruskalWallis), 112 18 (10.9%, n
4, p 0.001, KruskalWallis), 55 4 (5.4%, n 3, p 0.001,
KruskalWallis), and 28 9 (2.7%, n 4, p 0.001, Kruskal
Wallis), respectively. To confirm whether DTA-induced cell
death induction was restricted to MCH neurons, orexin/hypocretin neurons were stained and counted (Fig. 5 F, G). Orexin
neurons are distributed in the lateral hypothalamic area but do
not colocalize with MCH (Broberger et al., 1998; Elias et al., 1998;
Peyron et al., 1998; Bayer et al., 2002). The number of orexin
Discussion
In the present study, newly developed transgenic mouse strains
enabled manipulation of the activity and fate of MCH neurons in
vivo. The results obtained support a role for MCH neurons in the
regulation of both REM and NREM sleep.
Acute and reversible manipulation of MCH neuronal activity
using optogenetics
A role for MCH neurons in sleep/wake regulation has previously
been suggested based on studies using gene knock-out mice and
pharmacological studies. Prepro-MCH gene knock-out mice exhibited longer wake bouts during the dark period but REM sleep
was unchanged (Willie et al., 2008). MCHR1 gene knock-out
mice increased wakefulness and decreased NREM sleep (Ahnaou
et al., 2011). MCHR1 antagonists decreased deep NREM and
REM sleep quantities primarily by reducing the mean episode
duration (Ahnaou et al., 2008). MCH neurons are silent during
Figure 5. Ablation of MCH neurons increases wakefulness and decreases NREM sleep in MCH-tTA; TetO DTA bigenic mice. A, Schematic showing generation of bigenic MCH-tTA; TetO DTA mice. P,
Minimal promoter. B, Schematic illustration of the tetracycline-controlled gene expression system and tTA-induced DTA expression in MCH neurons. In the presence of Dox, Dox binds tTA protein and
DTA expression is repressed. C, Schematic shows the experimental protocol. In the presence of Dox, DTA expression is suppressed and MCH neurons are intact. Green bar, Chow contained Dox
[Dox()]. Open bar, Chow without Dox [Dox()]. D, Immunohistochemistry indicates MCH neuron-specific ablation in Dox(); 0, 1, 2, and 4 weeks (wk) (Figure legend continues.)
Other studies indicate that REM sleep is generated by glutamatergic neurons in the SLD that are specifically active during REM
sleep (Sakai and Koyama, 1996; Boissard et al., 2002; Lu et al.,
2006; Clement et al., 2011). These studies also suggest that MCH
neurons inhibit GABAergic REM-off neurons located in the ventrolateral periaqueductal gray region during REM sleep and that
these GABAergic REM-off neurons gate the activation of the
REM-on glutamatergic neurons in the SLD. MCH neurons might
function to open this gate to initiate REM sleep during NREM
sleep. This hypothesis is supported by our results following intermittent activation of MCH neurons. Although activation of
MCH neurons during NREM sleep induced REM, activation
during wakefulness had no effect on either REM or NREM sleep.
Direct transitions from wakefulness to REM sleep do not occur under normal conditions, but it occurs in narcoleptics in
whom orexin signaling pathways are interrupted. This observation indicates that, in contrast to MCH, orexin inhibits the initiation of REM sleep. Indeed, local administration of orexin into
the locus ceruleus or the laterodorsal tegmental nucleus dramatically suppresses REM sleep without affecting NREM sleep quantity (Bourgin et al., 2000; Xi et al., 2001). Orexin neurons and
MCH neurons are coextensive in the LHA, which leads us to
imagine a functional interaction between these neurons in the
regulation of sleep/wakefulness. Our observation that MCH neuron activation during NREM sleep induces REM sleep with
higher probability in the light period compared with the dark
period might reflect this interaction. Orexin concentrations in
the CSF are higher during the dark period compared with the
light period (Yoshida et al., 2001), which might interfere with the
transition from NREM to REM sleep when MCH neurons are
activated.
Chronic and irreversible ablation of MCH neurons
To further elucidate the role of MCH neurons in sleep/wakefulness, MCH neuron-specific ablation was performed. Nearcomplete MCH neuron ablation increased the total time in
wakefulness with decreased NREM sleep. However, the total time
in REM sleep was not affected. These observations indicate that
MCH neurons also play a crucial role in the regulation of NREM
sleep. The result from this chronic disruption of MCH neurotransmission conflicted with our results demonstrating that
acute activation of MCH neurons increases total time in REM
sleep. How could such different results between chronic ablation
and acute activation of MCH neurons arise? One possible explanation is suggested by a slice patch-clamp study that revealed that
MCH exhibited an inhibitory effect on orexin neurons (Rao et al.,
2008). The orexin and MCH systems often display opposing effects on physiological functions. For example, MCH knock-out
mice were hyperactive during fasting (Willie et al., 2008), whereas
orexin neuron-ablated mice were hypoactive but did not increase
locomotor activity during fasting (Hara et al., 2001; Yamanaka et
al., 2003). Orexin neurons are thought to be active during wakefulness and silent during REM sleep (Lee et al., 2005; Mileykovskiy et al., 2005; Hassani et al., 2009), whereas MCH neurons are
active in REM sleep and inactive during wakefulness (Hassani et
al., 2009). Putative somasomatic, axosomatic, and axodendritic
contacts between orexin and MCH neurons have been observed
(Bayer et al., 2002; Guan et al., 2002). Additionally, MCHR1
knock-out mice showed an increase in glutamatergic transmission onto orexin neurons. These facts support the concept that
MCH neurons have a role in the inhibition of orexin neuronal
activity. Therefore, ablation of MCH neurons might result in a
chronic activation of orexin neurons, which might cause an in-
crease in the total wake time and decrease in NREM sleep in MCH
neuron-ablated mice. However, acute inhibition of MCH neurons affected neither the time in wakefulness nor NREM sleep
duration. Optogenetic inhibition for 3 h might not be enough to
affect orexin neurons. Alternatively, after ablation of MCH neurons, neural network reorganization might occur, resulting in
chronic activation of orexin neurons.
MCH neurons also project to forebrain areas, such as the hippocampus and cortex (Bittencourt et al., 1992; Saito et al., 1999;
Hervieu et al., 2000). High levels of MCHR1 mRNA and MCHR1
immunoreactivity have been found in these areas (Bittencourt et
al., 1992; Saito et al., 1999; Hervieu et al., 2000). Therefore, in
addition to its role in sleep/wakefulness regulation, the release of
MCH onto cortical and hippocampal cells during REM sleep
might modulate long-term plasticity and contribute to the processing of learning and memory. Similar to the result of Konadhode et al., the increase of delta and theta power during activation
of MCH neurons might suggest the role of MCH on the cortex
(Konadhode et al., 2013). Additional studies are also required to
determine the role of MCH release during REM sleep in these
brain areas.
Together, the present results show that MCH neurons in the
hypothalamus play a significant role in the regulation of both
NREM and REM sleep. Further experiments are necessary to
identify the specific neural pathways that underlie the differential
roles that MCH neurons play in the regulation of NREM versus
REM sleep. The creation of MCH-tTA; TetO DTA mice, which
enable partial lesions of the MCH neuron population to be produced by varying the duration of Dox removal from the diet, may
be a useful tool in this regard.
Notes
Supplemental material for this article is available at https://dl.
dropboxusercontent.com/u/32320004/MCH-REM.mov. This material
has not been peer reviewed.
References
Ahnaou A, Drinkenburg WH, Bouwknecht JA, Alcazar J, Steckler T, Dautzenberg FM (2008) Blocking melanin-concentrating hormone MCH1
receptor affects rat sleep-wake architecture. Eur J Pharmacol 579:177
188. CrossRef Medline
Ahnaou A, Dautzenberg FM, Huysmans H, Steckler T, Drinkenburg WH
(2011) Contribution of melanin-concentrating hormone (MCH1) receptor to thermoregulation and sleep stabilization: evidence from MCH1
(/) mice. Behav Brain Res 218:4250. CrossRef Medline
Asakawa A, Inui A, Goto K, Yuzuriha H, Takimoto Y, Inui T, Katsuura G,
Fujino MA, Meguid MM, Kasuga M (2002) Effects of agouti-related
protein, orexin and melanin-concentrating hormone on oxygen consumption in mice. Int J Mol Med 10:523525. Medline
Bayer L, Mairet-Coello G, Risold PY, Griffond B (2002) Orexin/hypocretin
neurons: chemical phenotype and possible interactions with melaninconcentrating hormone neurons. Regul Pept 104:3339. Medline
Berndt A, Schoenenberger P, Mattis J, Tye KM, Deisseroth K, Hegemann P,
Oertner TG (2011) High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Proc Natl Acad Sci U S A 108:7595
7600. CrossRef Medline
Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, Vale W,
Sawchenko PE (1992) The melanin-concentrating hormone system of
the rat brain: an immuno- and hybridization histochemical characterization. J Comp Neurol 319:218 245. CrossRef Medline
Boissard R, Gervasoni D, Schmidt MH, Barbagli B, Fort P, Luppi PH
(2002) The rat ponto-medullary network responsible for paradoxical
sleep onset and maintenance: a combined microinjection and functional neuroanatomical study. Eur J Neurosci 16:1959 1973. CrossRef
Medline
Bourgin P, Huitron-Resendiz S, Spier AD, Fabre V, Morte B, Criado JR,
Sutcliffe JG, Henriksen SJ, de Lecea L (2000) Hypocretin-1 modulates